The contribution of immune regulatory microRNAs in endometriosis

Abstract

Endometriosis, the growth of ectopic endometrial tissue, affects 10% of reproductive-aged women. Although disease aetiology is enigmatic, aberrant immune responses within the peritoneal cavity have been implicated. Endometriotic lesion development is broadly classified into two phases; one mediated by a M1-like pro-inflammatory (tissue clearance; which inhibits disease) response and the other a M2-like anti-inflammatory (tissue remodelling; which promotes disease) response. Dysregulated expression of microRNA-155 (miR-155) in plasma samples from women with endometriosis is proposed to promote M1-like macrophage activity. Conversely, elevated miR-223 activity in endometriotic lesions is thought to promote M2-like macrophage activity. Hence, the aims of this doctoral thesis are to initially characterise endometriotic lesion development in an induced ‘menstrual’ mouse model of endometriosis, and to subsequently evaluate the impact of depleting either miR-155 or miR-223 on endometriotic-like lesion development and macrophage activity. Using the ‘menstrual’ mouse model of endometriosis, 40mg of decidualised donor endometrium was injected subcutaneously into recipient mice. Lesions that developed from syngeneic transfers (donor and recipient mice of the same genotype) and reciprocal transfers (between wildtype C57 mice and either miR-155-/- or miR-223-/- mice) were assessed by histochemical and immunohistochemical analysis for macrophage activity, angiogenesis and the extent of fibrosis at day (D)7, D14 and D21 after tissue transfer. To investigate effects on donor tissue gene expression, the differentially expressed genes (DEGs) and molecular pathways associated with the pathogenesis of endometriosis were defined by RNA-Sequencing of donor endometrium, as well as D7 and D14 lesions from syngeneic transfers using the Illumina Next-Seq500 platform at a depth of 50 million reads per samples. Differentially regulated genes were defined as those with a log fold change (log2FC) of 1 < log2FC <-1, and a false discovery rate (FDR) of 0.05. A reduction in lesion weight and size was seen over time in all groups. Effects of microRNA deficiency were seen on lesion tissue architecture, with an increase in glandular formation only evident in C57 to C57 and C57 to miR-155-/- transfers. Systemic deficiency of miR-155 acted to restrict M1-like macrophage activity and promoted the expression of M2-like macrophage markers. Importantly, blood vessel density increased in miR-155-/- to miR-155-/- lesions over time, supporting lesion establishment. In contrast, early influx of F4/80+ macrophages with increased MHC II and iNOS expression was seen in miR-223-/- to miR-223-/- lesions, associated with the development of cystic-like lesions devoid of glands. Similarly, by D14, glands were absent in lesions from C57 to miR-223-/- transfers. RNA-Seq analysis identified DEGs in several pathways associated with endometriosis, notably immune regulatory pathways, tissue remodelling, cellular differentiation and proliferation, and angiogenesis. In summary, this study has shown that microRNA modulation of macrophage polarisation impacts the development of endometriosis-like lesions in a mouse model, with deficiency in miR-155 increasing M2-like macrophage activity, while deficiency in miR-223 promotes M1-like macrophage activity. Reciprocal transfer data suggest that microRNA-dependent signalling factors from both the donor tissue and recipient environment influence macrophage activity, and have effects on endometriotic lesion growth. Our findings add to emerging evidence that macrophage phenotype and function are important determinants of endometriosis, and identify miR-155 and miR-223 as key microRNAs that regulate macrophage capacity to impact disease establishment and progression. Further studies are now required to determine whether similar microRNA-mediated modulation of macrophages contributes to human disease. In a clinical setting, the targeting of these macrophage-regulating microRNAs may have therapeutic potential, and should be investigated further.